Recombinant influenza virus vector carrying foreign genes in NA segment and preparation method and application thereof

Abstract

The invention discloses a recombinant influenza virus vector carrying foreign genes in an NA segment, which can be rescued in cells by a virus rescue method and can be amplified in chick embryos in large quantities. The invention also discloses a preparation method thereof, comprising the following steps: firstly constructing plasmids used for rescuing influenza viruses and introduced with the foreign genes on the NA genome segment of the influenza virus and carrying out rescue on host cells. The foreign gene is connected with the NA genome segment by a 2A segment. The foreign gene comprises EGFP reporter genes and adopts a CMV / huPolI bidirectional influenza virus rescue system. The recombinant influenza virus vector overcomes capacity limit of the foreign genes in the influenza virus genome, enjoys stable passage in chick embryos, can be applied to vaccine and drug development and tumor treatment and can produce recombinant cell factor drugs in cheap chick embryo bioreactors.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant influenza virus carrying a foreign gene as a virus vector and its preparation method and application. Background technique [0002] Viral vectors have been widely used in gene therapy research and vaccine development, and as effective gene delivery methods, they provide a powerful tool for basic research on life processes. The currently developed viral vectors are mainly DNA viruses, including adenovirus (Adenovirus, AdV), poxvirus (vacciniavirus), herpes virus (Herpes simple virus, HSV) and adeno-associated virus (Adeno-associated virus) vectors. , AAV). Although the lentiviral vector is an RNA viral vector, the functioning form of the lentiviral vector is the DNA form integrated into the host cell, and can also be classified as a DNA viral vector. There is a risk of integration of exogenous genes in the form of DNA in DNA virus vectors into the host genome, thus aff...

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Problems solved by technology

In order to carry exogenous genes, there have been attempts to carry exogenous genes by introducing influenza subgenome fragments, but the genes carried are small and unstable (seeing the following documents: Vieira Machado, A., Naffakh, N., Gerbaud, S. , van der Werf, S., and Escriou, N. (2006). Recombinant influenza A viruses harboring optimized dicistronic NA segment with an extended native 5′terminal sequence: Induction of heterospecific B and T cell responses in mice. Virology 345(1), 73-87; the Chinese name of the literature is that the recombinant influenza A virus with an extended 5' end region can carry optimized Shuangshun rice in the NA segment, and induces heterospecific B and T cell responses in mice)

There are also attempts to use vesicular stomatitis virus virus surface glycoprotein (VSV-G) to replace the HA and NA proteins of influenza virus in order to obtain a recombinant influenza virus that NA can carry foreign genes, but the recombinant influenza virus cannot be passed on chicken embryos , the cost of cultivating and amplifying this influenza virus on special types of cells is relatively high (see the following literature: Shinya, K., Fujii, Y., Ito, H., Ito, T., and Kawaoka, Y. (2004 ).Characterization of aneuraminidase-deficient influenza A virus as a potential gene delivery vector and alive vaccine.Journal of Virology 78(6), 3083-3088; research on gene delivery vectors and live vaccines)

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